Zh. Xu et al., Human 3 '-phosphoadenosine 5 '-phosphosulfate synthetase: Radiochemical enzymatic assay, biochemical properties, and hepatic variation, DRUG META D, 29(2), 2001, pp. 172-178
Sulfation is a major pathway in the biotransformation of many drugs and oth
er xenobiotic compounds. The sulfotransferase (SULT) enzymes that catalyze
these reactions use 3'-phosphoadenosine 5'-phosphosulfate (PAPS) as a sulfa
te donor cosubstrate. The synthesis of PAPS from inorganic sulfate and ATP
is catalyzed by PAPS synthetase (PAPSS). We previously cloned the genes for
human PAPSS1 and PAPSS2 as a step toward pharmacogenetic studies of these
enzymes. We have now developed a sensitive PAPSS radiochemical enzymatic as
say for use in genotype-phenotype correlation analyses. This coupled assay
uses the sulfation of 17 beta-[H-3] estradiol catalyzed by recombinant huma
n SULT1E1 to measure PAPS, which has been generated by PAPSS during the ini
tial step of the assay. SULT1E1 proved to be ideal for this application bot
h because of its relative resistance to inhibition by ATP, a substrate for
the PAPSS-catalyzed step, and because of its low K-m values for both PAPS (
58 nM) and estradiol (29 nM). After optimal PAPSS assay conditions had been
established, substrate kinetic studies were performed with cytosol prepara
tions from human liver and cerebral cortex, two tissues with very different
expression patterns for PAPSS1 and PAPSS2 mRNA. Brain and liver cytosol PA
PSS activities had apparent K-m values for ATP of 0.26 and 0.62 mM, respect
ively, and for SO42- of 0.08 and 0.31 mM, respectively. PAPSS activity was
then measured in 83 human liver biopsy samples to determine the nature and
extent of individual variation in this enzyme activity. An 18-fold variatio
n was observed. This sensitive new radiochemical assay can now be used in p
harmacogenetic studies of PAPSS in humans.